RNA reprogramming for RNA sensing and imaging in live cells
Embargo Date
2028-05-28
OA Version
Citation
Abstract
RNA plays central roles in diverse cellular processes, including gene expression and regulation at multiple levels. However, commonly used live-cell RNA imaging tools often require genetic modification of endogenous targets or exogenous probe delivery that complicates the detection process. Technologies that specifically and robustly sense RNA in its native biological context are thus highly desirable. This dissertation develops two genetically encoded RNA-based biosensors capable of RNA sensing in live prokaryotic and eukaryotic cells, respectively. First, I present tSENSE (tRNA-Scaffolded Engineered Nucleic acid Sensor), a class of programmable RNA switches that leverage conditional formation of tRNA scaffolds to improve fluorescence output in live E. coli. I demonstrate tSENSE design generalizability to accommodate diverse fluorogenic aptamer structures, programmability for dual-color sensing, and robustness in detecting functional RNA targets. Second, I introduce STAMP (Switchable Tandem Aptamer Probe), a class of de novo designed RNA switches that modulate tandem MS2 aptamer structure to enable sequence-specific RNA sensing in live HEK293T cells. I demonstrate both ON and OFF switch configurations with robust fold changes and apply STAMP ON switches to detect exogenously expressed mRNA. Together, these two platforms provide distinct and broadly applicable design principles for engineering genetically encoded RNA biosensors, expanding the toolkit for monitoring RNA dynamics in living cells.
Description
2026
License
Attribution-NonCommercial 4.0 International